EP1208091B1

EP1208091B1 - Benzophenones as inhibitors of reverse transcriptase

Info

Publication number: EP1208091B1
Application number: EP00967637A
Authority: EP
Inventors: Clarence Webster Glaxo Wellcome Inc. ANDREWS; Joseph Howing Glaxo Wellcome Inc CHAN; George Andrew Glaxo Wellcome Inc. FREEMAN; Karen Rene Glaxo Wellcome Inc. ROMINES; Jeffrey H. Glaxo Wellcome Inc. TIDWELL; Pascal Maurice Charles Pianetti
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 1999-09-04
Filing date: 2000-08-31
Publication date: 2006-05-03
Anticipated expiration: 2020-08-31
Also published as: CZ2002807A3; HUP0202593A2; NO323156B1; ES2261242T3; US7273863B1; CA2383782A1; HUP0202593A3; AU770302C; EP1208091A1; AR033344A1; DE60027729D1; JP2003510252A; JP3739704B2; AU7774300A; IL148322A0; PL354760A1; NO20021042D0; WO2001017982A1; AU770302B2; CN1391565A

Abstract

The present invention includes benzophenone compounds (I) which are useful in the treatment of HIV infections.

Description

Background of the Invention

The human immunodeficiency virus ("HIV") is the causative agent for acquired immunodeficiency syndrome ("AIDS"), a disease characterized by the destruction of the immune system, particularly of CD4⁺ T-cells, with attendant susceptibility to opportunistic infections, and its precursor AIDS-related complex ("ARC"), a syndrome characterized by symptoms such as persistent generalized lymphadenopathy, fever and weight loss. HIV is a retrovirus; the conversion of its RNA to DNA is accomplished through the action of the enzyme reverse transcriptase. Compounds that inhibit the function of reverse transcriptase inhibit replication of HIV in infected cells. Such compounds are useful in the prevention or treatment of HIV infection in humans.
Non-nucleoside reverse transcriptase inhibitors (NNRTIs), in addition to the nucleoside reverse transcriptase inhibitors gained a definitive place in the treatment of HIV-1 infections. The NNRTIs interact with a specific site of HIV-1 reverse transcriptase that is closely associated with, but distinct from, the NNRTI binding site. NNRTIs, however, are notorious for rapidly eliciting resistance due to mutations of the amino acids surrounding the NNRTI-binding site (E. De Clercq, II Famaco 54, 26-45, 1999). Failure of long-term efficacy of NNRTIs is often associated with the emergence of drug-resistant virus strains (J. Balzarini, Biochemical Pharmacology, Vol 58, 1-27, 1999). Moreover, the mutations that appear in the reverse transcriptase enzyme frequently result in a decreased sensitivity to other reverse transcriptase inhibitors, which results in cross-resistance.
JP 59181246 disclosed certain benzophenones useful as anticancer agents. Certain benzophenone derivatives as inhibitors of HIV-1 reverse bransriptase were disclosed in Wyatt et al. (J. Med. Chem. 38:1657-1665, 1995). However, these compounds were primarily active against wild-type HIV-1 reverse transcriptase, rapidly induced resistant virus, and were inactive against a common resistant strain.
We have now discovered that the compounds of the present invention are useful as inhibitors of both wild type and mutant variants of HIV reverse transcriptase.

Brief Description of the Invention

A first aspect of the invention features a compound selected from N [4-(aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3-chloro-5-cyanobenzoyl)phenoxy]acetamide and pharmaceutically acceptable derivatives thereof. These compounds are useful in the inhibition of HIV reverse transcriptase, particularly its resistant varieties, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment of AIDS and/or ARC, either as compounds, pharmaceutically acceptable salts or pharmaceutical composition ingredients. A second aspect of the invention features uses of the above-mentioned compounds in the manufacture of a medicament for treating AIDS, for preventing infection by HIV or for treating infection by HIV as monotherapy or in combination with other antivirals, anti-infectives, immunomodulators, antibiotics or vaccines. A third aspect of the invention features pharmaceutical compositions comprising the above-mentioned compounds and which are suitable for the prevention or treatment of HIV infection.

Detailed Description of the Invention

The present invention relates to a compound selected from N-[4-(aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3-chloro-5-cyanobenzoyl)phenoxy]acetamide; and pharmaceutically acceptable derivatives thereof in the inhibition of HIV reverse transcriptase and its resistant varieties, the prevention or treatment of infection by HIV and in the treatment of the resulting acquired immune deficiency syndrome (AIDS).
The term "pharmaceutically effective amount" refers to an amount effective in treating a virus infection, for example an HIV infection, in a patient either as monotherapy or in combination with other agents. The term "treating" as used herein refers to the alleviation of symptoms of a particular disorder in a patient, or the improvement of an ascertainable measurement associated with a particular disorder, and may include the suppression of symptom recurrence in an asymptomatic patient such as a patient in whom a viral infection has become latent. The term "prophylactically effective amount" refers to an amount effective in preventing a virus infection, for example an HIV infection, or preventing the occurrence of symptoms of such an infection, in a patient. As used herein, the term "patient" refers to a mammal, including a human.
The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the antiviral agent.
As used herein, the compounds according to the invention are defined to include pharmaceutically acceptable derivatives thereof. A "pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention or an inhibitorily active metabolite or residue thereof. Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.
Pharmaceutically acceptable salts of the compounds according to the invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicyclic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.
Salts derived from appropriate bases include alkali metal (e.g. sodium), alkaline earth metal (e.g., magnesium), ammonium and NW₄ ⁺ (wherein W is C_1-4 alkyl). Physiologically acceptable salts of a hydrogen atom or an amino group include salts of organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids. Physiologically acceptable salts of a compound with a hydroxy group include the anion of said compound in combination with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄ ⁺ (wherein W is a C_1-4alkyl group).
Esters of the compounds according to the invention are independently selected from the following groups: (1) carboxylic acid esters obtained by esterification of the hydroxy groups, in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, acetyl, n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted by, for example, halogen, C_1-4alkyl, or C_1-4alkoxy or amino); (2) sulfonate esters, such as alkyl- or aralkylsulfonyl (for example, methanesulfonyl); (3) amino acid esters (for example, L-valyl or L-isoleucyl); (4) phosphonate esters and (5) mono-, di- or triphosphate esters. The phosphate esters may be further esterified by, for example, a C_1-20 alcohol or reactive derivative thereof, or by a 2,3-di (C_6-24)acyl glycerol.
In such esters, unless otherwise specified, any alkyl moiety present advantageously contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Any cycloalkyl moiety present in such esters advantageously contains from 3 to 6 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group.
Any reference to any of the above compounds also includes a reference to a pharmaceutically acceptable salts thereof.
The compounds according to the invention can be used in medical therapy particularly for the treatment or prophylaxis of viral infections such as an HIV infection. Compounds according to the invention have been shown to be active against HIV infections, although these compounds may be active against HBV infections as well.
The compounds according to the invention are particularly suited to the treatment or prophylaxis of HIV infections and associated conditions. Reference herein to treatment extends to prophylaxis as well as the treatment of established infections, symptoms, and associated clinical conditions such as AIDS related complex (ARC), Kaposi's sarcoma, and AIDS dementia.
In particular, the compounds of the present invention may be used to treat wild-type HIV-1 as well as several resistance mutations, for example, K103N, L1001, or Y181C.
The compounds according to the invention may also be used in adjuvant therapy in the treatment of HIV infections or HIV-associated symptoms or effects, for example Kaposi's sarcoma.
In yet a further aspect, the present invention provides the use of a compound according to the invention in the manufacture of a medicament for the treatment or prophylaxis of any of the above mentioned viral infections or conditions.
The above compounds according to the invention and their pharmaceutically acceptable derivatives may be employed in combination with other therapeutic agents for the treatment of the above infections or conditions. Combination therapies according to the present invention comprise the administration of at least one compound of the present invention or a pharmaceutically acceptable derivative thereof and at least one other pharmaceutically active ingredient. The active ingredient(s) and pharmaceutically active agents may be administered simultaneously in either the same or different pharmaceutical formulations or sequentially in any order. The amounts of the active ingredient(s) and pharmaceutically active agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect. Preferably the combination therapy involves the administration of one compound according to the invention and one of the agents mentioned herein below.
Examples of such further therapeutic agents include agents that are effective for the treatment of viral infections or associated conditions such as (1 alpha, 2 beta, 3 alpha)-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine [(-)BHCG, SQ-34514], oxetanocin-G (3,4-bis-(hydroxymethyl)-2-oxetanosyl]guanine), acyclic nucleosides (e.g. acyclovir, valaciclovir, famcicolvir, ganciclovir, penciclovir), acyclic nucleoside phosphonates (e.g. (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine (HPMPC), PMEA, ribonucleotide reductase inhibitors such as 2-acetylpyridine 5-[(2-chloroanilino)thiocarbonyl) thiocarbonohydrazone, 3'azido-3'-deoxythymidine, other 2',3'-dideoxynucleosides such as 2',3'-dideoxycytidine, 2',3'-dideoxyadenosine, 2',3'-dideoxynosine, 2',3'-didehydrothymidine, protease inhibitors such as indinavir, ritonavir, nelfinavir, amprenavir, oxathiolane nucleoside analogues such as (-)-cis-1-(2-hydroxymethyl)-1,3-oxathiolane 5-yl)-cytosine (lamivudine) or cis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine (FTC), 3'-deoxy-3'-fluorothymidine, 5-chloro-2',3'-dideoxy-3' -fluorouridine, (-)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol (abacavir), ribavirin, 9-[4-hydroxy-2-(hydroxymethyl)but-1-yl]-guanine (H2G), tat inhibitors such as 7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2-(H)one (Ro5-3335), 7-chloro-1,3-dihydro-5-(1H-pyrrol-2yl)-3H-1,4-benzodiazepin-2-amine (Ro24-7429), interferons such as α-interferon, renal excretion inhibitors such as probenecid, nucleoside transport inhibitors such as dipyridamole; pentoxifylline, N-acetylcysteine (NAC), Procysteine, α-trichosanthin, phosphonoformic acid, as well as immunomodulators such as interleukin II or thymosin, granulocyte macrophage colony stimulating factors, erythropoetin, soluble CD4 and genetically engineered derivatives thereof, or other non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as nevirapine (BI-RG-587), loviride (α-APA) and delavuridine (BHAP), and phosphonoformic acid, and 1,4-dihydro-2H-3,1-benzoxazin-2-ones NNRTIs such as (-)-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one (L-743,726 or DMP-266), and quinoxaline NNRTIs such as isopropyl (2S)-7-fluoro-3,4-dihydro-2-ethyl-3-oxo-1(2H)-quinoxalinecarboxylate (HBY 1293).
The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
More preferably the combination therapy involves the administration of one of the above mentioned agents and a compound within one of the preferred or particularly preferred sub-groups within formulae (I) - (IV) (including IA, IB, IC and ID) as described above. Most preferably the combination therapy involves the joint use of one of the above named agents together with one of the compounds of the present invention specifically named herein.
The present invention further includes the use of a compound according to the invention in the manufacture of a medicament for simultaneous or sequential administration with at least one other therapeutic agent, such as those defined hereinbefore.
The compounds of the present invention may be synthesized by the following methods or by any method known in the art.
The compounds of the present invention may be prepared according to representative Schemes which are presented below. These schemes include also the preparation of other benzophenone derivatives as well as intermediates thereof not included in the scope of the claims. The compounds, which may be prepared according to these schemes, are not limited by the compounds contained in the schemes or by any particular substituents used in the schemes for illustrative purposes.
For example, carboxylic acid 49 (Scheme I) is allowed to react with amine 399 in DMF and in the presence of EDAC and HOBt at ambient temperature to provide compound 46.
For example, carboxylic acid 71 (Scheme II) is allowed to react with oxalyl chloride in dichloromethane and in the presence of a catalytic amount of DMF to afford the corresponding acid chloride. The acid chloride is then allowed to react with amine 466 in a mixture of acetone and water and in the presence of an excess of sodium bicarbonate to provide compound 78.
For example, phenol 4 (Scheme III) is allowed to react with 2'-chloroacetanilide in the presence of sodium carbonate in refluxing acetone to provide compound 1.
For example, phenol 47(Scheme IV) is allowed to react with ethyl bromoacetate in refluxing acetone and in the presence of potassium carbonate to afford ester 48.
For example, ester 48 (Scheme V) is allowed to react with lithium hydroxide in a mixture of THF, water, and ethanol to afford carboxylic acid 49.
For example, 2-bromo-4-chloroanisole (Scheme VI) in diethyl ether is treated with n-butyl lithium at -78 °C. After 15 minutes at -78 °C, the resulting lithium species is allowed to react with amide 68 to afford the desired ketone 69.
For example, 1-methyl-2-pyrrolecarboxylic acid (Scheme VII) in dichloromethane is allowed to react with excess oxalyl chloride in the presence of a catalytic amount of DMF. The resulting acid chloride is not isolated in pure form, but instead is allowed to react with N,O-dimethylhydroxylamine in chloroform and in the presence of triethylamine, to afford amide 14.
For example, 2-bromo-4-chloroanisole was treated with n-butyl lithium in ether and at -78 °C. The resulting lithio species is then allowed to react with 2-thiazolecarboxaldehyde to afford intermediate alcohol 2. Alcohol 2 is then allowed to react with an excess of manganese dioxide in dichloromethane at room temperature to afford ketone 3
For example, ketone 69 (Scheme IX) is allowed to react with an excess of boron tribromide in dichloromethane at -78°C to afford phenol 70.
For example, 4-chloroanisole (Scheme X) is allowed to react with 3,5-difluorobenzoyl chloride in refluxing dichloromethane in the presence of aluminum chloride to afford ketone 47.
For example, ester 223 (Scheme XI) is allowed to react with trimethylsilylacetytene, in the presence of tetrakis(triphenylphosphine)palladium, triethylamine and copper (I) iodide, to afford the intermediate trimethylsilyl-protected product. Treatment of the intermediate with tetrabutylammonium fluoride in THF provides compound 224
For example, compound 465 (Scheme XII) is allowed to react with 1N aqueous hydrochloric acid solution in ethanol at reflux temperature to afford 466.
For example, sulfonyl chloride 464 (Scheme XIII) is allowed to react with ammonium hydroxide in THF at ambient temperature to afford sulfonamide 465.
For example, compound 463 (Scheme XIV) is allowed to react with thionyl chloride in DMF at 0 °C to provide sulfonyl chloride 464.
For example, 2-aminotoluene-5-sulfonic acid (Scheme XV) is allowed to react with acetic anhydride in pyridine at ambient temperature to provide compound 462.
For example, compound 397 (Scheme XVI) is allowed to react with palladium on carbon in combination with hydrogen gas in ethyl alcohol at ambient temperature to afford compound 399.
For example, compound 394 (Scheme XVII) is allowed to react with MCPBA in chloroform at room temperature to provide both the sulfoxide 397 and the sulfone 398.
Two examples are shown below in Scheme XIX. In the first example, 5-fluoro-2-nitrotoluene is allowed to react with thiomorpholine in pyridine and water and in the presence of potassium carbonate to afford compound X. In the second example, 5-fluoro-2-nitrotoluene is allowed to react with imidazole in dimethylsulfoxide, in the presence of potassium carbonate, at 70 °C to provide compound 394.
The desired heterocycles, such as those used in the schemes above, are either commercially available or can be prepared using literature methods familiar to those skilled in the art.
For example, compound 139 (Scheme XX) is allowed to react with palladium on carbon in ethyl alcohol and in the presence of pressurized hydrogen gas to afford amine 140.
For example, 4-nitro-3-methylphenol (Scheme XXI) is allowed to react with 1,3-dibromopropane in DMF and in the presence of potassium carbonate to afford compound 249.
For example, sulfonyl chloride 260 (Scheme XXII) is allowed to react with dimethylamine in dichloromethane at 0 °C to provide sulfonamide 264.
For example, compound 253 (Scheme XXIII) is allowed to react with thionyl chloride in DMF at 0°C to afford sulfonyl chloride 254.
For example, 3-methyl-4-nitrophenol (Scheme XXIV) is allowed to react with 1,3-propane sultone in THF and in the presence of sodium hydride to afford sulfonic acid salt 253.
The desired sultones, such as 1,3-propane sultone, are either commercially available or can be prepared by literature methods familiar to those skilled in the art.
For example, 5-fluoro-2-nitrotoluene (Scheme XXV) is allowed to react with 4-(3-aminopropyl)morpholine in pyridine and water and in the presence of sodium bicarbonate to provide compound 308.
For example, 5-amino-4-methyl-2-pyridinesulfonamide can be prepared from 2-chloro-4-methyl-5-nitropyridine as shown in scheme XXVI. Commercially available 2-chloro-4-methyl-5-nitropyridine is allowed to react with an agent capable of displacing the 2-chloro group with a sulfur atom to provide 4-methyl-5-nitro-2-pyridinethiol, for example, thiourea. These reactions are typically performed in a polar, protic solvent, acetic acid, for example and in the presence of a base, potassium and sodium hydroxide for example, and at temperatures from 20 °C to 150 °C. The resulting thiol is then allowed to react with a reagent capable of oxidizing the thiol to the sulfonic acid derivative, for example hydrogen peroxide, oxone or chlorine gas. The oxidation can be advantageously performed using chlorine gas as the oxidizing agent in an acidic solvent, 1N hydrochloric acid for example, with the concomitant formation of the corresponding, desired sulfonyl chloride. The resulting sulfonyl chloride is then allowed to react with an agent capable of converting it to the corresponding sulfonamide, ammonia gas or a solution of ammonia in an appropriate solvent such as dichloromethane, to provide 4-methyl-5-nitro-2-pyridinesulfonamide. The nitro group can then be reduced using methods known to those skilled in the art, palladium on carbon in the presence of hydrogen gas as the reducing agent for example, to produce the desired 5-amino-4-methyl-2-pyridinesulfonamide. The reduction reactions are typically performed in a polar, protic solvent, methanol for example, and at temperatures from 20 °C to 100 °C, preferably at ambient temperature.
For example, 5-amino-2-methyl-3-nitropyridine was allowed to react with tert-butylnitrite, to produce the corresponding diazonium salt, followed by reaction with trimethylsilyl chloride in an aprotic solvent, dichloromethane for example, to afford 5-chloro-2-methyl-3-nitropyridine. The chloro group is then allowed to react with an agent capable of effecting a substitution on the pyridine ring to produce the corresponding thiol derivative. For example, 5-chloro-2-methyl-3-nitropyridine was allowed to react with thiourea in a mixture of acetic acid, potassium hydroxide and sodium hydroxide to afford the desired 6-methyl-5-nitro-2-pyridinethiol. The resulting thiol is then allowed to react with a reagent capable of oxidizing the thiol to the sulfonic acid derivative, for example hydrogen peroxide, oxone or chlorine gas. The oxidation can be advantageously performed using chlorine gas as the oxidizing agent in an acidic solvent, 1N hydrochloric acid for example, with the concomitant formation of the corresponding, desired sulfonyl chloride. The resulting sulfonyl chloride is then allowed to react with an agent capable of converting it to the corresponding sulfonamide, ammonia gas or a solution of ammonia in an appropriate solvent such as dichloromethane, to provide 6-methyl-5-nitro-2-pyridinesulfonamide. The nitro group can then be reduced using methods known to those skilled in the art, palladium on carbon in the presence of hydrogen gas as the reducing agent for example, to produce the desired 5-amino-6-methyl-2-pyridinesulfonamide. The reduction reactions are typically performed in a polar, protic solvent, methanol for example, and at temperatures from 20 °C to 100 °C, preferably at ambient temperature.
For example, 6-amino-5-methyl-3-pyridinesulfonamide can be prepared as shown in scheme XXVIII. Commercially available 2-amino-3-methylpyridine is allowed to react with an agent capable of sulfonylating the pyridine ring, for example oleum. These reactions are typically performed in a mixture of 20%SO₃/H₂SO₄, at temperatures ranging from 75 °C to 200 °C, preferably 160°C, to produce 6-amino-5-methyl-3-pyridinesulfonic acid. The amino group is then allowed to react with a combination of agents capable of effecting protection of the amino group from oxidation in subsequent steps. For example, 6-amino-5-methyl-3-pyridinesulonic acid was allowed to react with a mixture of N,N-dimethylformamide (DMF) and thionyl chloride, so-called Vilsmier reagents, to produce the desired 6-[(dimethylamino)methylidene]amino-5-methyl-3-pyridinesulfonic acid intermediate. This compound is then allowed to react with a combination of agents capable of converting the sulfonic acid to the corresponding sulfonyl chloride, followed by reaction with an agent capable of converting the sulfonyl chloride to the corresponding sulfonamide derivative. For example, desired 6-[(dimethylamino)methylidene]amino-5-methyl-3-pyridinesulfonic acid is allowed to react with phosphorous oxychloride to produce the intermediate sulfonyl chloride, followed by reaction with ammonium hydroxide, to afford the desired 6-amino-5-methyl-3-pyridinesulfonamide.
For example, 4-amino-N,3-dimethylbenzenesulfonamide can be prepared from commercially available 4-amino-3-methylbenzenesulfonic acid by reaction with a combination of reagents capable of effecting protection of the amino group from oxidation in later chemical steps. For example, 4-amino-3-methylbenzenesulfonic acid was allowed to react with N,N-dimethylformamide (DMF) and oxalyl chloride in dichloromethane to effect the concomitant protection of the amino group as the corresponding amidine as well as converting the sulfonic acid to the desired sulfonyl chloride. The sulfonyl chloride was then allowed to react with an amine, methyl amine for example, to produce 4-[(dimethylamino)methylidene]amino-N,3-dimethylbenzenesulfonamide. The amidine-protecting group was then removed using hydrazine hydrochloride.
For example, 4-amino-N,N,3-trimethylbenzenesulfonamide can be prepared by methods known in the art or as shown in Scheme XXX. Commercially available 4-amino-3-methylbenzenesulfonic acid is allowed to react with an agent capable of effecting protection of the amino group from oxidation in further synthetic steps. For example, 4-amino-3-methylbenzenesulfonic acid was allowed to react with benzyl bromide in the presence of a base, sodium or potassium carbonate for example, to afford sodium 4-(dibenzylamino)-3-metliylbenzenesulfonate. These reactions are typically perfonned a polar, aprotic solvent, N,N-dimethylformamide for example, at temperature ranges from 25°C to 125 °C, preferably 75-100 °C. The sodium salt is then allowed to react with an agent capable of converting the salt to the corresponding sulfonyl chloride. For example, sodium 4-(dibenzylamino)-3-methylbenzenesulfonate was allowed to react with thionyl chloride in N,N-dimethylformamide (DMF) to afford the desired 4-(dibenzylamino)-3-methylbenzenesulfonyl chloride. These reactions are typically performed in an aprotic solvent, dichloromethane for example, and at temperatures from 0°C to 75°C, preferably 0°C. The sulfonyl chloride is then allowed to react with an appropriate amine to afford the desired sulfonamide. For example, 4-(dibenzylamino)-3-methylbenzenesulfonyl chloride was allowed to react with dimethylamine to afford the desired 4-(dibenzylamino)-N,N,3-trimethylbenzenesulfonamide. The sulfonamide is then allowed to react with a combination of agents capable of effecting the deprotection of the amine to produce the desired aniline derivative. For example, desired 4-(dibenzylamino)-N,N,3-trimethylbenzenesulfonamide was allowed to react with hydrogen gas in the presence of a palladium on carbon catalyst to effect cleavage of the benzyl protecting groups and afford the desired 4-amino-N,N,3-trimethylbenzenesulfonamide.
For example, 3-methoxythiophene (Scheme XXXI) is allowed to react with benzoyl chloride in refluxing dichloromethane in the presence of aluminum chloride to afford ketone 664.
For example, 3,5-dibromotoluene in diethyl ether was treated with n-butyllithium at -78 °C. After 15 minutes at -78 °C, the resulting lithium species is allowed to react with 675 to afford the desired ketone 676 (see Scheme XXXIII).
See also Synthesis, 1984, 847 for the synthesis of 673 which after hydrolysis provided compound 674 (Scheme XXXIV).
The compounds according to the invention, also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and intravitreal). It will be appreciated that the preferred route will vary with the condition and age of the recipient, the nature of the infection and the chosen active ingredient.
In general a suitable dose for each of the above-mentioned conditions will be in the range of 0.01 to 250 mg per kilogram body weight of the recipient (e.g. a human) per day, preferably in the range of 0.1 to 100 mg per kilogram body weight per day and most preferably in the range 0.5 to 30 mg per kilogram body weight per day and particularly in the range 1.0 to 20 mg per kilogram body weight per day. Unless otherwise indicated, all weights of active ingredient are calculated as the parent compound of formula (I); for salts or esters thereof, the weights would be increased proportionally. The desired dose may be presented as one, two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. In some cases the desired dose may be given on alternative days. These sub-doses may be administered in unit dosage forms, for example, containing 10 to 1000 mg or 50 to 500 mg, preferably 20 to 500 mg, and most preferably 100 to 400 mg of active ingredient per unit dosage form.
While it is possible for the active ingredient to be administered alone it is preferable to present it as a pharmaceutical formulation. The formulations of the present invention comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof and optionally other therapeutic agents. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
Formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and intravitreal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods represent a further feature of the present invention and include the step of bringing into association the active ingredients with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
The present invention further includes a pharmaceutical formulation as hereinbefore defined wherein a compound of formula (I) or a pharmaceutically acceptable derivative thereof and at least one further therapeutic agent are presented separately from one another as a kit of parts.
Compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches suitably contain the active compound 1) in an optionally buffered, aqueous solution or 2) dissolved and/or dispersed in an adhesive or 3) dispersed in a polymer. A suitable concentration of the active compound is about 1% to 25%, preferably about 3% to 15%. As one particular possibility, the active compound may be delivered from the patch by electrotransport or iontophoresis as generally described in Pharmaceutical Research 3 (6), 318 (1986).
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, caplets, cachets or tablets each containing a predetermined amount of the active ingredients; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredients therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredients in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of the active combination with the softened or melted carrier(s) followed by chilling and shaping in molds.
Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents; and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or daily subdose of the active ingredients, as hereinbefore recited, or an appropriate fraction thereof.
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents.
The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way. "Active ingredient" denotes a compound according to the invention or multiples thereof or a physiologically functional derivative of any of the aforementioned compounds.

General Procedures:

General procedure II: Alkylation of phenols with ethyl bromoacetate

Into a round-bottom flask equipped with a stir bar, reflux condenser, and nitrogen on demand were placed the appropriate phenol, potassium carbonate (2-10 mmol/mmol of phenol), ethyl bromoacetate (1-1.5 mmol/mmol of phenol) and acetone (1-10 mL/mmol of phenol). The resulting mixture was heated to reflux for 1-20 h, after which time it was allowed to cool to rt and was poured into a separatory funnel containing ethyl acetate and water. The organic layer was collected and was washed with water, brine, dried over MgSO₄, filtered and the solvents were removed under reduced pressure to leave an oil. See specific examples for details regarding additional purification.

General procedure III: Saponification of ethyl esters to the carboxylic acids

A round-bottom flask was equipped with a stir bar, nitrogen on demand and was flushed with nitrogen. To the flask were added tetrahydrofuran (THF, 1-5 mL/mmol of ester), ethyl alcohol (EtOH, 1-5 mL/mmol of ester), water (1-5 mL/mmol of ester) and lithium hydroxide monohydrate (1-5 mmol/mmol of ester). The resulting suspension was stirred vigorously and the ester was added in one portion. The mixture was allowed to stir at rt for 1-20 h, after which time the pH was adjusted to approximately pH 5 by the slow addition of 1 N aqueous hydrochloric acid. The mixture was then poured into a separatory funnel containing ethyl acetate and water. The organic layer was collected and was washed with water, brine, dried over MgSO₄, filtered and the solvents were removed under reduced pressure to leave a white solid. See specific examples to determine if further purification of the product was required.

General Procedure V: Synthesis of acid chlorides from carboxylic acids using oxalyl chloride

Into a round-bottom flask were placed the appropriate carboxylic acid, methylene chloride (CH₂Cl₂, 1-10 mL/mmol acid), and N,N-dimethylformamide (1-10 drops). The mixture was cooled to 0 °C and oxalyl chloride (1-2 mmol/mmol acid) was added dropwise, after which time the mixture was allowed to warm to rt and stir for 1-24 h. The solvents were then removed under reduced pressure and the remaining residue was dried in vacuo. In most cases, the acid chlorides were used immediately used in subsequent reactions with no further purification.

General procedure VI: Coupling of acid chlorides to aromatic amines using sodium bicarbonate

Into a round-bottom flask were placed the appropriate aromatic amine, acetone (1-10 mL/mmol amine), sodium bicarbonate (2-10 mmol/mmol amine), and water (0.25-10 mL). The acid chloride was added as a solution in acetone (1-10 mL/mmol of acid chloride) in a dropwise manner and the reaction mixture was allowed to stir at rt for 1-24 h. When judged to be complete, the mixture was poured into a separatory funnel containing ethyl acetate and water. The organic layer was collected and was washed with water, brine, dried over MgSO₄, filtered and the solvents were removed under reduced pressure. See specific examples for details regarding further purification of the products.

General procedure VII: Synthesis of Weinreb amides from acid chlorides using N,O-dimethylhydroxylamine hydrochloride

Into a round bottom flask equipped with a stir bar and nitrogen on demand were placed the N,O-dimethylhydroxylamine (1-2 mmol/mmol acid chloride) and chloroform (CHCl₃, 1-10 mL/mmol acid chloride). The mixture was cooled to 0 °C and triethylamine (Et₃N, 1-5 mmol/mmol acid chloride) was added in one portion. The acid chloride was added and the reaction mixture was allowed to stir at 0 °C for 0.5-5 h, after which time was poured into a separatory funnel containing chloroform and water. The organics were collected, washed with water and brine, dried over MgSO₄, filtered and the solvents were removed under reduced pressure. See specific examples to determine if further purification of the product was required.

General procedure IX: Deprotection of anisole derivatives using boron tribromide

To a round-bottom flask equipped with a stir bar, nitrogen on demand, and an addition funnel was added the appropriate anisole derivative and methylene chloride (CH₂Cl₂, 1-15 mL/mmol of anisole). The mixture was cooled to -78 °C and boron tribromide was added dropwise at -78 °C. The resulting mixture was allowed to stir at -78 °C for 30-120 minutes, after which time it was allowed to warm to rt and stir for an additional 15-120 minutes. When judged to be complete, the reaction was poured over ice and extracted with CH₂Cl₂. The organics were collected, washed with water, dried over MgSO₄, filtered, and the solvents were removed. See specific examples to determine if further purification was required.

Reference Example 24

Step A:

Into a round-bottom flask equipped with a stir bar and nitrogen on demand were placed N,O-dimethylhydroxylamine hydrochloride (2.80 g, 28.7 mmol), Et₃N (9.0 mL, 64.57 mmol) and CHCl₃ (50 mL). The solution was cooled to 0°C and 3-trifluoromethyl-5-fluorobenzoyl chloride (5.0 g, 22.07 mmol) was added dropwise over several minutes. The resulting solution was allowed to stir at 0°C for 30 min, after which time it was allowed to warm to rt and stir for an additional 30 min. The mixture was then poured into a separatory funnel containing ethyl acetate and water. The organic layer was collected and was washed with water, brine, dried over MgSO₄, filtered and the solvents were removed under reduced pressure to provide 68 as a clear oil which was used without any further purification. ¹H NMR (CDCl₃, 300 MHz) δ 7.83 (s, 1H), 7.65 (d, J= 9 Hz, 1H), 7.46 (d, J= 9 Hz, 1H), 3.59 (s, 3H), 3.42 (s, 3H).

Step B:

Into a round-bottom flask equipped with a stir bar and nitrogen on demand were placed 2-bromo-4-chloroanisole (4.05 g, 18.29 mmol) and Et₂O (75 mL). The solution was cooled to -78°C and n-butyl lithium (13 mL of a 1.6 M solution in hexane, 20.8 mmol) was added dropwise. The resulting mixture was allowed to stir at -78°C for 15 min, after which time amide 68 (5.04 g, 20.07 mmol) was added dropwise. The mixture was allowed to stir at - 78°C for 30 min, after which time it was allowed to warm to rt and stir for an additional 2 h. The mixture was then poured into a separatory funnel containing ethyl acetate and water. The organic layer was collected and was washed with water, brine, dried over MgSO₄, filtered and the solvents were removed under reduced pressure afford 69 as a yellow solid (6.14 g, 92%), which was used in subsequent reactions without any further purification. ¹H NMR (CDCl₃, 300 MHz) δ 7.84 (s, 1H), 7.68 (d, J= 9 Hz, 1H), 7.58-7.51 (m, 2H), 7.44 (d, J= 3 Hz, 1H), 7.00 (d, J= 9 Hz, 1H), 3.74 (s, 3H).

Step C:

Into a round-bottom flask equipped with a stir bar and nitrogen on demand were placed 69 (6.14 g, 18.46 mmol) and CH₂Cl₂ (100 mL). The solution was cooled to -78°C and boron tribromide (50 mL of a 1.0 M solution in CH₂Cl₂, 50 mmol) was added dropwise over several minutes. The resulting dark mixture was allowed to stir at -78°C for 30 min, after which time it was allowed to warm to rt and stir for an additional 1h. The mixture was carefully poured over ice and the two-phase mixture was stirred for 30 min. It was then poured into a separatory funnel containing CH₂Cl₂ and water. The organic layer was collected, washed with water, brine, dried over MgSO₄, filtered and the solvents were removed under reduced pressure to afford 70 as a yellow solid (5.68 g, 96%), which was used without any further purification. ¹H NMR (CDCl₃, 300 MHz) δ 11.61 (s, 1H), 7.77 (s, 1H), 7.65-7.54 (m, 3H), 7.47 (d, J= 3 Hz, 1H), 7.12 (d, J= 9 Hz, 1H).

Step D:

Phenol 70 (5.68 g, 17.83 mmol), ethyl bromoacetate (2 mL, 18.03 mmol), K₂CO₃ (9.61 g, 69.53 mmol) and acetone (35 mL) were used according to general procedure II to provide the ester as a yellow, viscous oil which was used without any further purification. The ester (6.83 g, 16.88 mmol), lithium hydroxide (1.42 g, 33.84 mmol), water (20 mL), THF (50 mL) and EtOH (20 mL) were used according to general procedure III. The product was washed with several portions of ether to provide 71 as a white solid that was used without any further purification.

Reference Example 196

Carboxylic acid 71 (0.091 g, 0.24 mmol), methylene chloride (3 mL), DMF (4 drops), oxalyl chloride (0.057 mL, 0.65 mmol) were used as in general procedure V and added to a solution of 6-amino-1,1-dioxobenao(b)thiophene (Maybridge, 0.044 g, 0.24 mmol), acetone (1.0 mL), sodium bicarbonate (0.184 g, 2.2 mmol), and water (1 mL) as used in general procedure VI. The product was purified by filtering through a silica pad eluted with methylene chloride. The organics were washed with saturated sodium bicarbonate, dried over MgSO₄, and concentrated in vacuo. The product was further purified by flash chromatography using 9:1 methylene chloride:methanol as elutant to afford 461 as a yellow solid (0.013 g, 10%). ¹H NMR (DMSO-d₆, 400 MHz) δ4.75 (s, 2H), 7.2 (d, 1H), 7.25 (d, 1H), 7.5 (d, 1H), 7.54-7.58 (m, 2H), 7.59-7.64 (m, 2H), 7.85 (d, 2H), 7.9 (d, 1H), 8 (s, 1H), 10.4 (s, 1H); MS (ES^-) m/z 538 (M-H)^-.
Into a round-bottom flask were placed 2-aminotoluene-5-sulfonic acid (50.0 g, 267 mmol), and pyridine (300 mL). Acetic anhydride (38 mL, 403 mmol) was added dropwise from an addition funnel and the resulting mixture was allowed to stir for 2 h at rt. The solvents were removed under reduced pressure, to leave a brown solid. Several portions of ethyl alcohol were added to the solid and subsequently removed under reduced pressure, to afford a brown solid which was filtered and washed with several additional portions of ethyl alcohol and dried under vacuum (67.03 g, 81%) ¹H NMR (DMSO-d₆,) δ 2.08 (s, 3H), 2.22 (s, 3H), 7.39 (s, 2H), 7.45 (s, 1H), 8.02 (t, J= 6 Hz, 2H), 8.53 (t, J= 6Hz, 1H), 8.92 (d, J= 6Hz, 2H), 9.31 (s, 1H).
Compound 462 (67.03 g, 217 mmol) was added to a round-bottom flask containing 1N NaOH (225 mL) and the resulting mixture was allowed to stir at rt for 3 h. The mixture was concentrated under reduced pressure, to afford a brown solid. Several portions of ethyl alcohol were added and subsequently removed under reduced pressure. The remaining solid was filtered, washed with a final portion of ethyl alcohol and dried under vacuum (42.34 g, 77%). ¹H NMR (DMSO-d₆, 300 MHz) δ 2.08 (s, 3H), 2.22 (s, 3H), 7.39 (s, 2H), 7.45 (s, 1H), 9.31 (s, 1H).
Sulfonic acid salt 463 (42.34 g, 169 mmol) and DMF (300 mL) were added to a flask that was equipped with a stir bar and nitrogen on demand and was cooled to 0 °C. Thionyl chloride (30 mL, 411 mmol) was added dropwise from an addition funnel at a rate such that the temperature of the reaction mixture did not exceed 10 °C. When the addition was complete, the mixture was allowed to warm to rt and stir for an additional 2 1/2 h, after which time it was poured into a beaker containing crushed ice. The resulting solid was collected by filtration, washed with several portions of water and dried under vacuum (25.63 g, 61%). ¹H NMR (DMSO, d₆, 400 MHz) δ 2.02 (s, 3H), 2.15 (s, 3H), 7.33 (s, 2H), 7.38 (s, 1H), 9.27 (s, 1H).
Into a round-bottom flask, equipped with a stir bar and nitrogen on demand, were placed sodium acetate (19.82 g, 241.6 mmol) and ethyl alcohol (200 mL) and the mixture was cooled to 0 °C. Ammonia gas was bubbled through the sodium acetate solution for 5 min, then sulfonyl chloride 464 (25.63 g, 103 mmol) was added as a solid and in one portion. The resulting mixture was allowed to stir at 0 °C for 30 min, and was then allowed to warm to rt and stir for an additional 18 h. The mixture was then diluted with water and was poured into a separatory funnel containing water and ethyl acetate. The organic layer was collected, washed with water, brine, dried over MgSO₄, filtered and the solvents were removed under reduced pressure to provide 465 as a yellow solid (8.4 g, 36%), which was used without further purification.
A round-bottom flask was equipped with a stir bar, a reflux condenser and nitrogen on demand. Into the flask were placed sulfonamide 465 (8.4 g, 36.80 mmol), ethyl alcohol (200 mL) and 2N hydrochloric acid (128 mL). The resulting mixture was allowed to heat to reflux overnight, after which time it was allowed to cool to RT and was neutralized with saturated, aqueous sodium bicarbonate. It was then poured into a separatory funnel containing water and ethyl acetate, the organic layer was collected, washed with water, brine, dried over MgSO₄, filtered and the solvents were removed under reduced pressure to afford a tan solid (6.35 g, 93%), which was used without further purification. ¹H NMR (DMSO-d₆, 400 MHz) δ 2.06 (s, 3H), 5.54 (s, 2H), 6.58 (d, J= 12 Hz, 1H), 6.82 (s, 2H), 7.30 (d, J= 12 Hz, 1H), 7.33 (s, 1H).

Reference Example 197:

Step A:

The title compound was prepared according to General Procedure VII from 3,5-dimethoxybenzoyl chloride (2.00 g, 10.0 mmol). The reaction gave 468 as a colorless oil (2.143 g, 95%): ¹H NMR (CDCl₃, 400 MHz) δ 6.75 (d, 2 H), 6.49 (t, 1H), 3.76 (s, 6 H), 3.55 (s, 3 H), 3.29 (s, 3 H).

Step B:

A solution of 2-bromo-4-chlorophenol (0.830 g, 4.0 mmol) in 20 mL of THF was cooled to -78 °C in a dry ice/acetone bath. n-Butyllithium (5.5 mL of a 1.6 M solution in hexanes, 8.8 mmol) was added dropwise over 5 min, and the resulting mixture was stirred at -78 °C for 1 h. A solution of 468 (0.901 g, 4.0 mmol) in 5 mL of THF was added dropwise over 4 min, and the resulting mixture was stirred at -78 °C for 1.25 h, then at room temperature for 14 h. The reaction mixture was poured into 50 mL of water and extracted with two 50-mL portions of EtOAc. The combined organic layers were then dried over MgSO₄, filtered and concentrated in vacuo to give 1.193 g of a brown oil. Purification by flash chromatography using 10% EtOAc/hexanes as an eluant followed by crystallization from hot ether gave 469 as yellow crystals (0.234 g, 20%): ¹H NMR (CDCl₃, 300 MHz) δ 11.83 (s, 1 H), 7.62 (d, 1 H), 7.45 (dd, 1 H), 7.03 (d, 1 H), 6.76 (d, 2 H), 6.68 (t, 1 H), 3.84 (s, 6 H).

Step C:

A solution of 466 (5.0 g, 26.85 mol) and pyridine (2.4 mL, 29.53 mmol) in 150 mL of chloroform was cooled to 0 °C in an ice bath. Bromoacetyl bromide (2.6 mL, 29.53 mmol) was added dropwise over 20 min, and the resulting mixture was allowed to slowly warm to room temperature as it was stirred for 18 h. The reaction mixture was then poured into 150 mL of water and extracted with two 100-mL portions of CH₂Cl₂. Both the organic and aqueous layers were filtered to yield a beige solid. This solid was suspended in 40 mL of 1 N HCl and stirred several minutes. The solid was then filtered and rinsed with CH₂Cl₂, MeOH, and hexanes to yield 470 (5.705 g, 69%): ¹H NMR (CDCl₃, 400 MHz) δ 9.84 (s, 1H), 7.66-7.56 (m, 3 H), 7.23 (br s, 2 H), 4.09 (s, 2 H), 2.24 (s, 3 H).

Step D:

A mixture of 469 (0.144 g, 0.49 mmol), 470 (0.162 g, 0.53 mmol), and potassium carbonate (0.339 g, 2.45 mmol) in 5 mL of acetone was warmed to reflux for 6 h, then stirred at room temperature overnight. The reaction mixture went dry overnight, so another 5 mL of acetone was added, and the resulting mixture was heated to reflux for 8 h, then stirred at room temperature for 22 h. The reaction mixture was poured into 30 mL of water and extracted with two 30-mL portions of EtOAc. The combined organic layers were filtered to remove solid, washed with brine, dried over MgSO₄, filtered, and concentrated in vacuo to give 0.195 g of a yellow solid. Purification by suspension in hot ether followed by filtration gave 467 (0.094 g, 37%): MS (AP+) m/z 518.9 (M+H); ¹H NMR (DMSO-d₆, 400 MHz) δ 9.15 (s, 1H), 7.63-7.60 (m, 3 H), 7.56 (dd, 1 H), 7.39 (d, 1 H), 7.22 (s, 2H), 7.18 (d, 1 H), 6.82 (d, 2 H), 6.71 (t, 1 H), 4.76 (s, 2 H), 3.69 (s, 6 H), 2.12 (s, 3 H).

Reference Example 198:

Step A:

A solution of 1,3,5-tribromobenzene (9.44 g, 30 mmol) in 120 mL of ether was cooled to -78 °C in a dry ice/acetone bath. n-Butyllithium (13.2 mL of 2.5 M solution in hexanes, 33 mmol) was added dropwise over 10 min. The resulting mixture was stirred at -78 °C for an additional 10 min, then hexachloroethane (7.15 g, 30.2 mmol) was added in small portions over 3 min. The reaction mixture was then stirred for 15 min at -78 °C, followed by 3.2 h at rt. The mixture was partitioned between 100 mL of water and 100 mL of EtOAc. The aqueous layer was separated and extracted with an additional 100 mL of EtOAc. The combined organic layers were then dried over MgSO₄ filtered, and concentrated in vacuo to give 472 as a pale brown solid (7.72 g, 95%): ¹H NMR (CDCl₃, 300 MHz) δ 7.57 (t, 1 H), 7.47 (d, 2 H).

Step B:

A solution of 472 (7.62 g, 28.2 mmol) in 100 mL of ether was cooled to -78 °C in a dry ice/acetone bath. n-Butyllithium (12.6 mL of 2.5 M solution in hexanes, 31.5 mmol) was added dropwise over 30 min. The resulting mixture was stirred at -78 °C for an additional 13 min, then 183 (6.57 g, 28.6 mmol) was added in small portions over 23 min. The reaction mixture was then stirred for 22 h as the bath was allowed to warm to room temperature. The mixture was poured into 100 mL water and extracted with two 100-mL portions of EtOAc. The combined organic layers were then dried over MgSO₄, filtered, and concentrated in vacuo to give 9.46 g of a beige solid. Recrystallization from hot MeOH gave 473 (6.45 g, 64%): MS (AP-) m/z 358 (M-H); ¹H NMR (CDCl₃, 300 MHz) δ 7.76 (t, 1 H), 7.70 (t, 1 H), 7.65 (t, 1 H), 7.47 (dd, 1 H), 7.36( d, 1 H), 6.95 (d, 1 H), 3.72 (s, 3 H).

Step C:

The title compound was prepared according to General Procedure IX from 473 (0.338 g, 0.94 mmol). The reaction gave 474 (0.325 g, 100%): ¹H NMR (CDCl₃, 400 MHz) δ 11.54 (s, 1 H), 7.72 (t, 1 H), 7.62 (d, 1 H), 7.52 (d, 1 H), 7.46 (dd, 1 H), 7.41 (d, 1 H), 7.02 (d, 1 H).

Step D:

A mixture of 474 (0.173 g, 0.50 mmol), 470 (0.154 g, 0.50 mmol), and potassium carbonate (0.346 g, 2.5 mmol) in 10 mL of acetone was warmed to reflux for 15 h and stirred at room temperature another 4 h. The reaction mixture was then poured into 35 mL of water and extracted with two 35-mL portions of EtOAc. The aqueous layer was then filtered and extracted with another 20 mL of EtOAc. The combined organic layers were washed with brine, dried over MgSO₄, filtered, and concentrated in vacuo to give 0.230 g of a yellow oil. Purification by flash chromatography using 0.5-1 % MeOH/CH₂Cl₂ gave 471 (0.048 g, 17%): MS (AP+) m/z 573 (M+H); ¹H NMR (DMSO-d ₆, 400 MHz) δ 9.36 (s, 1 H), 7.97 (t, 1 H), 7.79 (s, 1 H), 7.71 (s, 1 H), 7.68-7.47 (m, 4 H), 7.45 (d, 1 H), 7.21 (s, 2 H), 7.20-7.18 (d, 1 H), 4.77 (s, 2 H), 2.13 (s, 3 H).

Example 199:

Step A:

A solution of 473 (0.299 g, 0.83 mmol), sodium cyanide (0.086 g, 1.76 mmol), copper (I) iodide (0.028 g, 0.15 mmol), and tetrakis-(triphenylphosphine)-palladium (0.113 g, 0.10 mmol) in 8 mL of acetonotrile was heated to reflux for 40 min. The reaction mixture was then diluted with 50 mL of EtOAc and filtered through Celite. The resulting solution was washed with 25 mL of water, dried over MgSO₄, filtered and concentrated in vacuo to give 0.375 g of an orange gum. Purification by flash chromatography using 5% EtOAc/hexane as the eluant gave 476 (0.171 g, 56%): ¹H NMR (CDCl₃, 400 MHz) δ 7.93 (t, 1H), 7.82 (t, 1 H), 7.76 (t, 1 H), 7.47 (dd, 1 H), 7.37 (d, 1 H), 6.93 (d, 1 H), 3.67 (s, 3 H).

Step B:

The title compound was prepared according to General Procedure IX from 476 (0.165 g, 0.54 mmol). The reaction gave 477 (0.174 g, 100%): ¹H NMR (CDCl₃, 400 MHz) δ 11.43 (s, 1 H), 7.84-7.82 (m, 2 H), 7.78 (t, 1 H), 7.49 (dd, 1 H), 7.34 (d, 1 H), 7.05 (d, 1 H).

Step C:

A mixture of 477 (0.157 g, 0.54 mmol), 470 (0.165 g, 0.54 mmol), and potassium carbonate (0.373 g, 2.7 mmol) in 10 mL of acetone was warmed to reflux for 17.5 h. The reaction mixture was then poured into 35 mL of water and extracted with two 35-mL portions of EtOAc. The combined organic layers were washed with brine, dried over MgSO₄, filtered, and concentrated in vacuo to give 0.276 g of a yellow oil. Purification by flash chromatography using 0.5-1% MeOH/CH₂Cl₂ gave 475 (0.033 g, 12%): MS (AP-) mlz 517 (M-H); ¹H NMR (DMSO-d₆, 400 MHz) δ 9.42 (s, 1H), 8.26 (s, 1 H), 8.11 (s, 1 H), 8.03 (t, 1 H), 7.63 (dd, 1 H), 7.60-7.53 (m, 3 H), 7.49 (d, 1 H), 7.22 (s, 2 H), 7.19 (d, 1H), 4.77 (s, 2 H),2.14 (s, 3 H).

Inhibition of Viral Replication

I. HeLa Cell Assay

The HeLa cell assay was performed according to a modifcation of Kimpton J. and Emerman M., Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated β-galactosidase gene, J. Virol. 66:2232-2239 (1992), in which HIV-1 infection is detected by the activation of an HIV-LTR driven β-galactosidase reporter that is integrated into the genome of a CD4⁺ HeLa cell line. Quantitation of β-galactosidase is achieved by measuring the activation of a chemiluminescent substrate (Tropix). The concentration of each compound required to inhibit 50% (IC₅₀) of the HIV-1 induced β-galactosidase signal, relative to untreated controls, was determined for each isogenic, recombinant virus.

A. Experimental Procedure

Growth and Maintenance of the CD4-HIV LTR-β-gal HeLa cell line.

HeLa-CD4-LTR-β-gal cells were obtained from the NIH AIDS Research and Reference Reagent Program. Cells were propagated in DMEM containing 10% fetal bovine serum, 0.2 mg/ml geneticin and 0.1 mg/ml hygromycin B. Cells were routinely split by trypsinization when confluency reached 80% (approximately every 2 to 3 days).

B. Construction of HIV-1 reverse transcriptase (RT) mutants

DNA encoding the HIV-1 reverse transcriptase was subcloned from a M13 phage into a general shuttle vector, pBCSK+, as a ~1.65 kbp EcoRI/HindIII ended DNA fragment. The HIV DNA insert of the resulting plasmid, pRT2, was completely sequenced on both strands prior to use in site directed mutagenesis experiments. Specific amino acid replacements were made using Stratagene Quick Change reagents and mutagenic oligonucleotides from Oligos. Following mutagenesis, the entire mutant RT coding sequence was verified by sequencing both DNA strands.

C. Construction of isogenic HIV-1 RT mutant virus

Mutant HIV-1 strains were isolated by a modified Recombinant Virus Assay (Kellam P. and Larder B., Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates, Antimicrobial Agents and Chemotherapy, 38:23-30, 1994). 1 X 10⁷ Jurkat T-cells (maintained in RPMI containing 10% fetal bovine serum, split 1:5 every 5 to 6 days) were co-transfected with EcoRI/HindIII digested mutant RT plasmid and Bst EII-digested HIV-1_HXB2ΔRTDNA in the presence of DMRIE-C transfection reagent (Gibco) according to supplier's recommended protocol. Each mutant RT coding sequence was crossed into the RT-deleted HIV-1 viral DNA backbone by in vivo homologous recombination. Transfected cell cultures were expanded and monitored until syncytia formation and CPE were extensive. Virus was harvested by clear spin of the culture supernatants and frozen at - 80 C as primary stock. Recombinant progeny virus was sequenced in the RT region to confirm the mutant genotype. Virus stocks were further expanded by infection of Jurkat cells, harvested and stored as frozen aliquots. Stocks were titered in HeLa MAGI cells for assay.

D. Titering of virus stocks

The HIV-1_HXB2 mutants were titered in the HeLa MAGI assay system to determine the relative light units (RLU) per ml, a measure of infectivity relevant for this assay system. Virus stocks were diluted in a 2-fold series into DMEM containing 10% fetal bovine serum plus 20µg/ml DEAE-dextran and assayed as described in the Experimental Protocol section, below.

E. Experimental Protocol

96-well microtiter plate(s) (Costar #3598) were seeded with 3 X 10³ HeLa-CD4-LTR-β- gal in 100µl DMEM containing 10% fetal bovine serum. Plates were placed in a 37 °C, 5% CO₂ humidified incubator overnight. The following day, mutant virus stocks were thawed in a room temperature water bath and diluted into DMEM containing 10% fetal bovine serum and 20µg/ml DEAE-dextran to achieve an input of 1500 to 2000 RLU/ml. All media was removed with an 8 channel manifold aspirator and 35µl (50 to 70 total RLUs) of diluted virus was added to each well for virus adsorption. Plates were placed in a 37 °C, 5% CO₂ humidified incubator for 1.5 to 2 hours.
Compound titration plates were prepared at 1.35X final concentration during the virus adsorption period. Compounds were titrated robotically in a five-fold stepwise manner from 2.7 µM (2µM final) to 1.35 pM (1pM final). This scheme allows 8 compounds to be tested per 96-well plate with 10 dilution points and 2 controls per compound (n=1). Compounds were titrated into DMEM containing 10% fetal bovine serum plus 0.135% DMSO (0.1% final). 100µl of titrated compound was removed from every well of the titration plate and added to the virus adsorption plate. Plates were placed in a 37 °C, 5% CO₂ humidified incubator for 72 hours.
Following incubation, supernatants were aspirated from every well as described above and 100µl of phosphate buffered saline was added. The PBS was then aspirated as above and 15µl of lysis buffer (Tropix) was added. Plates were maintained at room temperature for 10 minutes during which time the chemiluminescent substrate (Tropix) was diluted 1:50 into room temperature substrate dilution buffer (Tropix). 100µl of diluted substrate was then added to each well. Plates were incubated at room temperature for 1 to 1.5 hours. Following incubation, the chemiluminescence of each well was measured with a Dynatech plate reader using the following settings:

PARAMETER VALUE

run cycle

data all

gain low

cycles 1s

pause 2s

rows abedefgh

temp room

stir off

The output raw data, RLUs, were analyzed by nonlinear regression to determine IC₅₀ values (see data analysis section below).

F. Data Analysis

Relative light units (RLU) are expressed as % control: $(RLU at compound [] / RLU no compound) * 100 = % Control$

The concentration of compound that inhibits 50% of the signal produced in untreated samples (IC₅₀) is determined by the following nonlinear regression model available on the ROBOSAGE software package: $Y = V_{\max} * (1 - (X^{n} / (K^{n} + X^{n})))$

This equation describes a sigmoidal inhibition curve with a zero baseline. X is inhibitor concentration and Y is the response being inhibited. V_max is the limiting response as X approaches zero. As X increases without bound, Y tends toward its lower limit, zero. K is the IC₅₀ for the inhibition curve, that is, Y is equal to 50% of V_max when X = K.
Results in Table 1 are reported as ranges of representative IC₅₀ values.

II. MT4 Cell Assay

A. Experimental Procedure

Antiviral HIV activity and compound-induced cytotoxicity were measured in parallel by means of a propidium iodide based procedure in the human T-cell lymphotropic virus transformed cell line MT4. Aliquots of the test compounds were serially diluted in medium (RPMI 1640, 10% fetal calf serum (FCS), and gentamycin) in 96-well plates (Costar 3598) using a Cetus Pro/Pette. Exponentially growing MT4 cells were harvested and centrifuged at 1000 rpm for 10 min in a Jouan centrifuge (model CR 4 12). Cell pellets were resuspended in fresh medium (RPMI 1640, 20% FCS, 20% IL-2, and gentamycin) to a density of 5 x 10 cells/ml. Cell aliquots were infected by the addition of HIV-1 (strain IIIB) diluted to give a viral multiplicity of infection of 100 x TCID50. A similar cell aliquot was diluted with medium to provide a mock-infected control. Cell infection was allowed to proceed for 1 hr at 37°C in a tissue culture incubator with humidified 5% CO₂ atmosphere. After the 1 hr incubation the virus/cell suspensions were diluted 6-fold with fresh medium, and 125 µl of the cell suspension was added to each well of the plate containing pre-diluted compound. Plates were then placed in a tissue culture incubator with humidified 5% CO₂ for 5 days. At the end of the incubation period, 27 µl of 5% Nonidet-40 was added to each well of the incubation plate. After thorough mixing with a Costar multitip pipetter, 60 µl of the mixture was transferred to filter-bottomed 96-well plates. The plates were analyzed in an automated assay instrument (Screen Machine, Idexx Laboratories). The control and standard used was 3'-azido-3'-deoxythymidine tested over a concentration range of 0.01 to 1 µM in every assay. The expected range of IC₅₀ values for 3'-azido-3'-deoxythymidine is 0.04 to 0.12 µM. The assay makes use of a propidium iodide dye to estimate the DNA content of each well.

B. Analysis

The antiviral effect of a test compound is reported as an IC₅₀, i.e. the inhibitory concentration that would produce a 50% decrease in the HIV-induced cytopathic effect. This effect is measured by the amount of test compound required to restore 50% of the cell growth of HIV-infected MT4 cells, compared to uninfected MT4 cell controls. IC₅₀ was calculated by RoboSage, Automated Curve Fitting Program, version 5.00, 10-Jul-1995.
For each assay plate, the results (relative fluorescence units, rfU) of wells containing uninfected cells or infected cells with no compound were averaged, respectively. For measurements of compound-induced cytotoxicty, results from wells containing various compound concentrations and uninfected cells were compared to the average of uninfected cells without compound treatment. Percent of cells remaining is determined by the following formula: $Percent of cells remaining = (compound-treated uninfected cell, rfU / untreated uninfected cells) \times 100.$
A level of percent of cells remaining of 79% or less indicates a significant level of direct compound-induced cytotoxicity for the compound at that concentration. When this condition occurs the results from the compound-treated infected wells at this concentration are not included in the calculation of IC₅₀.
For measurements of compound antiviral activity, results from wells containing various compound concentrations and infected cells are compared to the average of uninfected and infected cells without compound treatment. Percent inhibition of virus is determined by the following formula: $Percent inhibition of virus = (1 - ((ave . untreated uninfected cells - treated infected cells) / (ave . untreated uninfected cells - ave . untreated infected cells))) \times 100$

References:

1. Averett, D.R., Anti-HIV compound assessment by two novel high capacity assays, J. Virol. Methods 23: 263-276, 1989.
2. Schwartz, O., et al,. A rapid and simple colorimetric test for the study of anti-HIV agents, AIDS Res. and Human Retroviruses 4 (6): 441-447, 1988..
3. Daluge, S.M., et al., 5-chloro-2'3'-deoxy-3'fluorouridine (935U83), a selective anti-human immunodeficiency virus agent with an improved metabolic and toxicological profile. Antimicro. Agents and Chemother. 38 (7): 1590-1603, 1994.
4. Dornsife, R.E., et al., Anti-human immunodeficiency virus synergism by zidovudine (3'-azidothymidine) and didanosine (dideoxyinosine) contrasts with the additive inhibition of normal human marrow progenitor cells, Antimicro. Agents and Chemother. 35 (2): 322-328, 1991.

Results in Table 1. are expressed as representative IC₅₀ ranges.

Table 1

Compound Number	Virus Type	IC50 (nM) Range *	Assay
475	HIV-I	A	MT4
	NEV-R	A	MT4
	G190A	A	HeLa
	K103N	A	HeLa
	K103N/G190A	A	HeLa
	K103N/P225H	A	HeLa
	K103N/V1081	A	HeLa
	K103N/Y181C	A	HeLa
	L100I	A	HeLa
	P225H	A	HeLa
	P236L	A	HeLa
	V106A/Y181C	A	HeLa
	V1061	A	HeLa
	V1061/Y181C	B	HeLa
	V1081	A	HeLa
	V1081/Y181C	A	HeLa
	WTRVA	A	HeLa
	Y181C	A	HeLa

* A indicates an IC₅₀ of 10nM or less
B indicates an lC₅₀ between 11nM and 100nM
C indicates an IC₅₀ between 101nM and 1,000nM
D indicates an IC₅₀ between 1.000nM and 3,000nM

Claims

A compound selected from
N-[4-(aminosulfonyl)-2-methylphenyl]-2-[4-chloro-2-(3-chloro-5-cyanobenzoyl)phenoxy]acetamide;
and pharmaceutically acceptable salts, esters or salts of an ester thereof.
A compound according to claim 1 for use in medical therapy.
Use of a compound according to claim 1 in the manufacture of a medicament for the treatment of a viral infection.
The use according to claim 3 wherein the viral infection is an HIV infection.
A pharmaceutical composition comprising an effective amount of a compound according to claim 1 together with a pharmaceutically acceptable carrier.
A pharmaceutical composition according to claim 5 in the form of a tablet or capsule.
A pharmaceutical composition according to claim 5 in the form of a liquid.